Apoptosis

Apoptosis (pronounced ă-pŏp-tŏ’sĭs) is a form of programmed cell death in multicellular organisms. It is one of the main types of programmed cell death (PCD) and involves a series of biochemical events leading to a characteristic cell morphology and death, in more specific terms, a series of biochemical events that lead to a variety of morphological changes, including blebbing, changes to the cell membrane such as loss of membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation (1-4). Processes of disposal of cellular debris whose results do not damage the organism differentiates apoptosis from necrosis.




In contrast to necrosis, which is a form of traumatic cell death that results from acute cellular injury, apoptosis, in general, confers advantages during an organism's life cycle. For example, the differentiation of fingers and toes in a developing human embryo occurs because cells between the fingers apoptose; the result is that the digits are separate. Between 50 billion and 70 billion cells die each day due to apoptosis in the average human adult. For an average child between the ages of 8 and 14, approximately 20 billion to 30 billion cells die a day. In a year, this amounts to the proliferation and subsequent destruction of a mass of cells equal to an individual's body weight.


Research on apoptosis has increased substantially since the early 1990s. In addition to its importance as a biological phenomenon, defective apoptotic processes have been implicated in an extensive variety of diseases. Excessive apoptosis causes hypotrophy, such as in ischemic damage, whereas an insufficient amount results in uncontrolled cell proliferation, such as cancer.


Process


The process of apoptosis is controlled by a diverse range of cell signals, which may originate either extracellularly (extrinsic inducers) or intracellularly (intrinsic inducers). Extracellular signals may include hormones, growth factors, nitric oxide or cytokines, and therefore must either cross the plasma membrane or transduce to effect a response. These signals may positively or negatively induce apoptosis; in this context the binding and subsequent initiation of apoptosis by a molecule is termed positive, whereas the active repression of apoptosis by a molecule is termed negative.

Intracellular apoptotic signalling is a response initiated by a cell in response to stress, and may ultimately result in cell suicide. The binding of nuclear receptors by glucocorticoids, heat, radiation, nutrient deprivation, viral infection, and hypoxia are all factors that can lead to the release of intracellular apoptotic signals by a damaged cell. A number of cellular components, such as poly ADP ribose polymerase, may also help regulate apoptosis.

Before the actual process of cell death is carried out by enzymes, apoptotic signals must be connected to the actual death pathway by way of regulatory proteins. This step allows apoptotic signals to either culminate in cell death, or be aborted should the cell no longer need to die. Several proteins are involved, however two main methods of achieving regulation have been identified; targeting mitochondria functionality, or directly transducing the signal via adapter proteins to the apoptotic mechanisms. The whole preparation process requires energy and functioning cell machinery.


Mitochondrial regulation




The mitochondria are essential to multicellular life. Without them, a cell ceases to respire aerobically and quickly dies - a fact exploited by some apoptotic pathways. Apoptotic proteins that target mitochondria affect them in different ways; they may cause mitochondrial swelling through the formation of membrane pores, or they may increase the permeability of the mitochondrial membrane and cause apoptotic effectors to leak out.There is also a growing body of evidence that indicates that nitric oxide (NO) is able to induce apoptosis by helping to dissipate the membrane potential of mitochondria and therefore make it more permeable.

Mitochondrial proteins known as SMACs (second mitochondria-derived activator of caspases) are released into the cytosol following an increase in permeability. SMAC binds to inhibitor of apoptosis proteins (IAPs) and deactivates them, preventing the IAPs from arresting the apoptotic process and therefore allowing apoptosis to proceed. IAP also normally suppresses the activity of a group of cysteine proteases called caspases, which carry out the degradation of the cell, therefore the actual degradation enzymes can be seen to be indirectly regulated by mitochondrial permeability.

Cytochrome c is also released from mitochondria due to formation of a channel, MAC, in the outer mitochondrial membrane, and serves a regulatory function as it precedes morphological change associated with apoptosis. Once cytochrome c is released it binds with Apaf-1 and ATP, which then bind to pro-caspase-9 to create a protein complex known as an apoptosome. The apoptosome cleaves the pro-caspase to its active form of caspase-9, which in turn activates the effector caspase-3.

MAC is itself subject to regulation by various proteins, such as those encoded by the mammalian Bcl-2 family of anti-apoptopic genes, the homologs of the ced-9 gene found in C. elegans. Bcl-2 proteins are able to promote or inhibit apoptosis either by direct action on MAC or indirectly through other proteins. It is important to note that the actions of some Bcl-2 proteins are able to halt apoptosis even if cytochrome c has been released by the mitochondria.



Direct signal transduction


Two important examples of the direct initiation of apoptotic mechanisms in mammals include the TNF-induced (tumour necrosis factor) model and the Fas-Fas ligand-mediated model, both involving receptors of the TNF receptor (TNFR) family coupled to extrinsic signals.

TNF is a cytokine produced mainly by activated macrophages, and is the major extrinsic mediator of apoptosis. Most cells in the human body have two receptors for TNF: TNF-R1 and TNF-R2. The binding of TNF to TNF-R1 has been shown to initiate the pathway that leads to caspase activation via the intermediate membrane proteins TNF receptor-associated death domain (TRADD) and Fas-associated death domain protein (FADD). Binding of this receptor can also indirectly lead to the activation of transcription factors involved in cell survival and inflammatory responses.The link between TNF and apoptosis shows why an abnormal production of TNF plays a fundamental role in several human diseases, especially in autoimmune diseases.

The Fas receptor (also known as Apo-1 or CD95) binds the Fas ligand (FasL), a transmembrane protein part of the TNF family.The interaction between Fas and FasL results in the formation of the death-inducing signaling complex (DISC), which contains the FADD, caspase-8 and caspase-10. In some types of cells (type I), processed caspase-8 directly activates other members of the caspase family, and triggers the execution of apoptosis. In other types of cells (type II), the Fas-DISC starts a feedback loop that spirals into increasing release of pro-apoptotic factors from mitochondria and the amplified activation of caspase-8.

Following TNF-R1 and Fas activation in mammalian cells a balance between pro-apoptotic (BAX,BID, BAK, or BAD) and anti-apoptotic (Bcl-Xl and Bcl-2) members of the Bcl-2 family is established. This balance is the proportion of pro-apoptotic homodimers that form in the outer-membrane of the mitochondrion. The pro-apoptotic homodimers are required to make the mitochondrial membrane permeable for the release of caspase activators such as cytochrome c and SMAC. Control of pro-apoptotic proteins under normal cell conditions of non-apoptotic cells is incompletely understood, but it has been found that a mitochondrial outer-membrane protein, VDAC2, interacts with BAK to keep this potentially-lethal apoptotic effector under control.When the death signal is received, products of the activation cascade displace VDAC2 and BAK is able to be activated.



Execution


Although many pathways and signals lead to apoptosis, there is only one mechanism that actually causes the death of the cell in this process; after the appropriate stimulus has been received by the cell and the necessary controls exerted, a cell will undergo the organised degradation of cellular organelles by activated proteolytic caspases. A cell undergoing apoptosis shows a characteristic morphology that can be observed with a microscope:

1. Cell shrinkage and rounding due to the breakdown of the proteinaceous cytoskeleton by caspases.
2. The cytoplasm appears dense, and the organelles appear tightly packed.
3. Chromatin undergoes condensation into compact patches against the nuclear envelope in a process known as pyknosis, a hallmark of apoptosis.
4. The nuclear envelope becomes discontinuous and the DNA inside it is fragmented in a process referred to as karyorrhexis. The nucleus breaks into several discrete chromatin bodies or nucleosomal units due to the degradation of DNA.
5. The cell membrane shows irregular buds known as blebs.
6. The cell breaks apart into several vesicles called apoptotic bodies, which are then phagocytosed.


Apoptosis progresses quickly and its products are quickly removed, making it difficult to detect or visualize. During karyorrhexis, endonuclease activation leaves short DNA fragments, regularly spaced in size. These give a characteristic "laddered" appearance on agar gel after electrophoresis. Tests for DNA laddering differentiate apoptosis from ischemic or toxic cell death.



Removal of dead cells


Dying cells that undergo the final stages of apoptosis display phagocytotic molecules, such as phosphatidylserine, on their cell surface.Phosphatidylserine is normally found on the cytosolic surface of the plasma membrane, but is redistributed during apoptosis to the extracellular surface by a hypothetical protein known as scramblase. These molecules mark the cell for phagocytosis by cells possessing the appropriate receptors, such as macrophages.Upon recognition, the phagocyte reorganizes its cytoskeleton for engulfment of the cell. The removal of dying cells by phagocytes occurs in an orderly manner without eliciting an inflammatory response.

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Central Dogma Animation

The central dogma of molecular biology was first enunciated by Francis Crick in 1958 and re-stated in a Nature paper published in 1970
The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that such information cannot be transferred back from protein to either protein or nucleic acid.



In other words, 'once information gets into protein, it can't flow back to nucleic acid.'


The dogma is a framework for understanding the transfer of sequence information between sequential information-carrying biopolymers, in the most common or general case, in living organisms. There are 3 major classes of such biopolymers: DNA and RNA (both nucleic acids), and protein. There are 3×3 = 9 conceivable direct transfers of information that can occur between these. The dogma classes these into 3 groups of 3: 3 general transfers (believed to occur normally in most cells), 3 special transfers (known to occur, but only under abnormal conditions), and 3 unknown transfers (believed to never occur). The general transfers describe the normal flow of biological information: DNA can be copied to DNA (DNA replication), DNA information can be copied into mRNA, (transcription), and proteins can be synthesized using the information in mRNA as a template (translation).


Biological sequence information
Biopolymers are biological polymers. That is, they are molecules made up of building blocks known as monomers. The biopolymers DNA, RNA and proteins, are linear polymers (ie: each monomer connects to at most two other monomers). The sequence, or arrangement of their monomers, effectively encodes information. The transfers of information described by the central dogma are faithful, deterministic transfers, wherein one biopolymer's sequence is used as a template for the construction of another biopolymer with a sequence that is entirely dependent on the original biopolymer's



DNA Replication

As the final step in the Central Dogma, to transmit the genetic information between parents and progeny, the DNA must be replicated faithfully. Replication is carried out by a complex group of proteins that unwind the superhelix, unwind the double-stranded DNA helix, and, using DNA polymerase and its associated proteins, copy or replicate the master template itself so the cycle can repeat DNA → RNA → protein in a new generation of cells or organisms

Transcription
Transcription is the process by which the information contained in a section of DNA is transferred to a newly assembled piece of messenger RNA (mRNA). It is facilitated by RNA polymerase and transcription factors. In eukaryote cells the primary transcript (pre-mRNA) is often processed further via alternative splicing. In this process, blocks of mRNA are cut out and rearranged, to produce different arrangements of the original sequence.

Translation
Eventually, this mature mRNA finds its way to a ribosome, where it is translated. In prokaryotic cells, which have no nuclear compartment, the process of transcription and translation may be linked together. In eukaryotic cells, the site of transcription (the cell nucleus) is usually separated from the site of translation (the cytoplasm), so the mRNA must be transported out of the nucleus into the cytoplasm, where it can be bound by ribosomes. The mRNA is read by the ribosome as triplet codons, usually beginning with an AUG, or initiator methonine codon downstream of the ribosome binding site. Complexes of initiation factors and elongation factors bring aminoacylated transfer RNAs (tRNAs) into the ribosome-mRNA complex, matching the codon in the mRNA to the anti-codon in the tRNA, thereby adding the correct amino acid in the sequence encoding the gene. As the amino acids are linked into the growing peptide chain, they begin folding into the correct conformation. This folding continues until the nascent polypeptide chains are released from the ribosome as a mature protein. In some cases the new polypeptide chain requires additional processing to make a mature protein. The correct folding process is quite complex and may require other proteins, called chaperone proteins. Occasionally proteins themselves can be further spliced, when this happens the inside "discarded" section is known as an intein.



Special transfers of biological sequential information
Reverse transcription
Reverse transcription is the transfer of information from RNA to DNA (the reverse of normal transcription). This is known to occur in the case of retroviruses, such as HIV, and, in higher eukaryotes, in the case of retrotransposons. It is not, however, the general case in most living organisms.

RNA replication
RNA replication is the copying of one RNA to another. It is possible that this is the mechanism by which some RNA viruses replicate.

Direct translation from DNA to protein

Direct translation from DNA to protein has been demonstrated in a cell-free system (i.e. in a test tube), using extracts from E. Coli that contained ribosomes, but not intact cells. These cell fragments could express proteins from foreign DNA templates, and neomycin was found to enhance this effect

Methylation
Variation in methylation states of DNA can alter gene expression levels significantly. Methylation variation usually occurs through the action of DNA methylases (which are proteins). When the change is heritable, it is considered epigenetic. When the change in information status is not heritable, it would be a somatic epitype. The effective information content has been changed by means of the actions of a protein or proteins on DNA, but the primary DNA sequence is not altered.

Prions - almost an "unknown transfer"

Prions are proteins that propagate themselves by making conformational changes in other molecules of the same type of protein. This change affects the behaviour of the protein. In fungi this change can be passed from one generation to the next, i.e. Protein → Protein. Although this represents a transfer of information, it is not an exception to the central dogma, since the sequence of the protein remains unchanged.







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